Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells

ABSTRACT

Novel yeast cell transformation vectors are manufactured and employed in securing expression of exogenous polypeptides in yeast cells. Vectors include promoter/regulator DNA sequences of yeast glyceraldehyde-3-phosphate dehydrogenase gene origins. In an illustrative preferred embodiment, novel immunologically active hepatitis B surface antigen (HBsAg) preparations are isolated from yeast cells transformed with plasmid A.T.C.C. 40053. These HBsAg preparations of yeast origin may be incorporated into vaccine compositions useful in developing immunological responses protective against infection by hepatitis B virus.

This application is a continuation of application Ser. No. 748,712 filedJune 26, 1985, now abandoned, which is a continuation of application ofSer. No. 412,707 filed Aug. 30, 1982, now abandoned.

BACKGROUND

The present invention relates, in part, to manipulation of geneticmaterials including the manufacture of specific DNA sequences useful inrecombinant procedures to secure the production of proteins of interestby microorganisms. The present invention also relates to novelimmunologically active substances produced by recombinant methodologiesand, more particularly to novel preparations of hepatitis B surfaceantigen (HBsAg) of yeast cell origin.

A. Manipulation Of Genetic Materials

Genetic materials may be broadly defined as those chemical substanceswhich program for and guide the manufacture of constituents of cells andviruses and direct the responses of cells and viruses. A long chainpolymeric substance known as deoxyribonucleic acid (DNA) comprises thegenetic material of all living cells and viruses except for certainviruses which are programmed by ribonucleic acids (RNA). The repeatingunits in DNA polymers are four different nucleotides, each of whichconsists of either a purine (adenine or guanine) or a pyrimidine(thymine or cytosine) bound to a deoxyribose sugar to which a phosphategroup is attached. Attachment of nucleotides in linear polymeric form isby means of fusion of the 5' phosphate of one nucleotide to the 3'hydroxyl group of another. Functional DNA occurs in the form of stabledouble stranded associations of single strands of nucleotides (known asdeoxyoligonucleotides), which associations occur by means of hydrogenbonding between purine and pyrimidine bases [i.e., "complementary"associations existing either between adenine (A) and thymine (T) orguanine (G) and cytosine (C)]. By convention, nucleotides are referredto by the names of their constituent purine or pyrimidine bases, and thecomplementary associations of nucleotides in double stranded DNA (i.e.,A-T and G-C) are referred to as "base pairs". Ribonucleic acid is apolynucleotide comprising adenine, guanine, cytosine and uracil (U),rather than thymine, bound to ribose and a phosphate group.

Most briefly put, the programming function of DNA is generally effectedthrough a process wherein specific DNA nucleotide sequences (genes) are"transcribed" into relatively unstable messenger RNA (mRNA) polymers.The mRNA, in turn, serves as a template for the formation of structural,regulatory and catalytic proteins from amino acids. This mRNA"translation" process involves the operations of small RNA strands(t+RNA) which transport and align individual amino acids along the mRNAstrand to allow for formation of polypeptides in proper amino acidsequences. The mRNA "message", derived from DNA and providing the basisfor the tRNA supply and orientation of any given one of the twenty aminoacids for polypeptide "expression", is in the form of triplet"codons"--sequential groupings of three nucleotide bases. In one sense,the formation of a protein is the ultimate form of "expression" of theprogrammed genetic message provided by the nucleotide sequence of agene.

Certain DNA sequences which usually "precede" a gene in a DNA polymerprovide a site for initiation of the transcription into mRNA. These arereferred to as "promoter" sequences. Other DNA sequences also usually"upstream" of (i.e., preceding) a gene in a given DNA polymer, bindproteins that determine the frequency (or rate) of transcriptioninitiation. These other sequences are referred to as "regulator"sequences. Thus, sequences which precede a selected gene (or series ofgenes) in a functional DNA polymer and which operate to determinewhether the transcription (and eventual expression) of a gene will takeplace are collectively referred to as "promoter/regulator" or "control"DNA sequences. DNA sequences which "follow" a gene in a DNA polymer andprovide a signal for termination of the transcription into mRNA arereferred to as transcription "terminator" sequences.

A focus of microbiological processing for nearly the last decade hasbeen the attempt to manufacture industrially and pharmaceuticallysignificant substances using organisms which do not initially havegenetically coded information concerning the desired product included intheir DNA. Simply put, a gene that specifies the structure of a productis either isolated from a "donor" organism or chemically synthesized andthen stably introduced into another organism which is preferably aself-replicating unicellular microorganism. Once this is done, theexisting machinery for gene expression in the "transformed" host cellsoperates to construct the desired product.

The art is rich in patent and literature publications relating to"recombinant DNA" methodologies for the isolation, synthesis,purification and amplification of genetic materials for use in thetransformation of selected host organisms. U.S. Letters Patent No.4,237,224 to Cohen, et al., for example, relates to transformation ofprocaryotic unicellular host organisms with "hybrid" viral or circularplasmid DNA which includes selected exogenous DNA sequences. Theprocedures of the Cohen, et al. patent first involve manufacture of atransformation vector by enzymatically cleaving viral or circularplasmid DNA to form linear DNA strands. Selected foreign ("exogenous" orheterologous") DNA strands are also prepared in linear form through useof similar enzymes. The linear viral or plasmid DNA is incubated withthe foreign DNA in the presence of ligating enzymes capable of effectinga restoration process and "hybrid" vectors are formed which include theselected foreign DNA segment "spliced" into the viral or circular DNAplasmid.

Transformation of compatible unicellular host organisms with the hybridvector results in the formation of multiple copies of the foreign DNA inthe host cell population. In some instances, the desired result issimply the amplification of the foreign DNA and the "product" harvestedis DNA. More frequently, the goal of transformation is the expression bythe host cells of the foreign DNA in the form of large scale synthesisof isolatable quantities of commercially significant protein orpolypeptide fragments coded for by the foreign DNA. See also, e.g., U.S.Pat. Nos. 4,264,731 (to Shine), 4,273,875 (to Manis) and 4,293,652 (toCohen).

The success of procedures such as described in the Cohen, et al. patentis due in large part to the ready availability of "restrictionendonuclease" enyzmes which facilitate the site-specific cleavage ofboth the unhybridized DNA vector and, e.g., eucaryotic DNA strandscontaining the foreign sequences of interest. Cleavage in a mannerproviding for the formation of single stranded complementary "ends" onthe double stranded linear DNA strands greatly enhances the likelihoodof functional incorporation of the foreign DNA into the vector upon"ligating" enzyme treatment. A large number of such restrictionendonuclease enzymes are currently commercially available [See, e.g.,"BRL Restriction Endonuclease Reference Chart" appearing in the "'81/'82Catalog" of Bethesda Research Laboratories, Inc., Gaithersburg,Maryland.] Verification of hybrid formation is facilitated bychromatographic techniques which can, for example, distinguish thehybrid plasmids from non-hybrids on the basis of molecular weight. Otheruseful verification techniques involve radioactive DNA hybridization.

Another manipulative "tool" largely responsible for successes intransformation of procaryotic cells is the use of selectable "marker"gene sequences. Briefly put, hybrid vectors are employed which contain,in addition to the desired foreign DNA, one or more DNA sequences whichcode for expression of a phenotypic trait capable of distinguishingtransformed from non-transformed host cells. Typical marker genesequences are those which allow a transformed procaryotic cell tosurvive and propagate in a culture medium containing metals,antibiotics, and like components which would kill or severely inhibitpropagation of non-transformed host cells.

Successful expression of an exogenous gene in a transformed hostmicroorganism depends to a great extent on incorporation of the geneinto a transformation vector with a suitable promoter/regulator regionpresent to insure transcription of the gene into mRNA and other signalswhich insure translation of the mRNA message into protein (e.g.,ribosome binding sites). It is not often the case that the "original"promoter/regulator region of a gene will allow for high levels ofexpression in the new host. Consequently, the gene to be inserted musteither be fitted with a new, host-accommodated transcription andtranslation regulating DNA sequence prior to insertion or it must beinserted at a site where it will come under the control of existingtranscription and translation signals in the vector DNA.

Expression of Genes By Yeast Cells

Of particular interest to the background of the present invention ofnovel transformation vectors are those publications which provideinformation concerning the general nature of expression of polypeptidesin yeast cells (e.g., Saccharomyces cerevisiae) as well as those whichdescribe attempts to employ yeast cells as host cells for the expressionof exogenous (e.g., mammalian) genes which code for commercially usefulpolypeptides. Yeast cells comprise particularly interesting potentialhost cells for practice of recombinant method directed toward expressionof glycoproteins because the existing synthetic apparatus of these cellshas the capacity to generate glycosylated proteins.

Illustrative of those publications focusing on the general nature ofgene expression in yeast cells are those reporting on studies of theprimary structure of genes coding for synthesis of endogenous yeastenzymes. See, e.g., Holland and Holland, J. Biol. Chem., 254, pp.9839-9845 (1979) discussing the yeast glyceraldehyde-3-phosphatedehydrogenase (G-3-PDH) gene; Hitzeman, et al., J. Biol. Chem., 255, pp.12073-12080 (1980) discussing the yeast 3-phosphoglycero kinase (PGK)gene; and, Bennetzen and Hall, J. Biol. Chem., 257, pp. 3018-3025 (1982)discussing the yeast alcohol dehyrogenase I (ADHl) gene. Publicationstreating genes coding for polypeptides whose presence or absence canserve as metabolic markers for potential yeast cell transformationvectors include: Hinnen, et al., Proc. Natl. Acad. Sci. U.S.A., 75, pp.1929-1933 (1978) discussing the yeast LEU2 gene coding for the leucinebiosynthetic enzyme, β-isopropyl-malate dehydrogenase; and Tschumper andCarbon, Gene, 16, pp. 157-166 (1980) which discusses the yeast TRPl genewhich codes for the tryptophane biosynthetic enzyme phosphoribosylanthranilate isomerase.

A number of prior publications treat autonomous replicating DNAsequences, "ARS's", which confer the capacity for autonomous replicationof contiguous DNA sequences and are thus significantly involved ininsuring autonomous replication of DNA transformation vectors in yeastcells. Beggs, Nature, 275, pp. 104-109 (1979) discusses the so-called"2μ" origin of replication". See, also, Stinchcomb, et al., Proc. Natl.Acad. Sci. U.S.A., 77, pp. 4559 (1980) which treats DNA sequences ofnon-yeast origins which are capable of functioning as ARS's in yeast.Struhl, et al., Proc. Natl. Acad. Sci. U.S.A., 76, pp. 1035-1039 (1979)discusses the so-called "ARS 1" origin of replication sequence obtainedas a 1453 base pair fragment of yeast DNA upon digestion with therestriction endonuclease enzyme, EcoRI. Hsiao and Carbon, Proc. Natl.Acad. Sci. U.S.A., 76, pp. 3829-3833 reports the isolation of an ARS ona fragment containing the yeast ARG 4 gene.

The above-noted Struhl, et al. reference is of interest for itsdiscussion of the ARS 1 origin of replication in the context of thedevelopment of a particular kind of yeast transformation vector,yeast-bacterial hybrid plasmids. These plasmids are able to replicate in(and may be selected in and recovered from) both E. coli (wherein it ispreferred to effect vector construction and amplification) andSaccharomyces cerevisiae (wherein it may be desired to secure DNAexpression). The development and properties of such hybrid plasmids,commonly referred to as "shuttle vectors" has subsequently beenextensively treated in Stinchcomb, et al., Nature, 282, pp. 39-43(1979); Kingsman, et al., Gene, 7, pp. 141-153 (1979); and, Tschumperand Carbon, Gene, 10, pp. 157-166 (1980). These references specificallydiscuss the yeast bacterial plasmid designated YRp7.

Finally, the efficiency of termination of transcription of eucaryoticgenes, including yeast genes, into mRNA has been the subject of manyrecent studies. A summary of the more significant findings of suchstudies may be found in Zaret, et al., Cell, 28, pp. 563-573 (1982),which itself treats transcription efficiency for mutant and wild typeyeast CYCl genes. Briefly put, efficient transcription of yeast DNA intomRNA appears to be significantly dependent upon the presence or absenceof either a site for so-called "poly-A addition" (typically an AATAAAsequence), or a "transcription termination" sequence, or both suchsequences, at or near the 3' end of a polypeptide coding region of agene.

While the above-noted references have provided a great deal ofinformation which is relevant to the goal of securing expression ofexogenous genes in yeast cells, reports of successful, stable yeast celltransformations with accompanying high levels of expression of exogenousgenes are exceedingly few. For example, very recent publications havedealt with successful attempts to secure expression of human leucocyteinterferon D [Hitzeman, et al., Nature, 293, pp. 717-722 (1982)] andunsuccessful attempts to secure expression of rat growth hormone[Ammerer, et al., Recombinant DNA, Proc. 3rd Cleveland Symp.Macromolecules (ed. Walton, AG), pp. 185-197, (Elsevier Amsterdam,1981)]. In each instance, the work involved construction of yeastexpression vectors which combine a selectable marker (LEU2 or TRP1), ayeast replication origin (ARSl or the 2μ origin) as well as what aregenerically referred to as "transcription initiation andtermination-specifying sequences, primarily from the yeast ADHl gene".In each instance an intron-free gene of mammalian origin was inserted 3'to an ADHl promoter/regulator region fragment and 5' to a yeast TRPlgene, viz, 5'-ADHl promoter-3', 5'-mammalian gene-3',5'-TRPl-3'.

The "mixed" results of this research involving essentially identicalprocedures practiced by the same group of researchers but generatingsignificantly different results in terms of protein expression wererecently summarized in an abstract by Hall and Ammerer appearing in DNA,2, page 182 (1982) wherein it was noted, "By joining the 5' flankingsequences of the yeast ADHl structural gene to the coding sequences ofother genes (yeast CYCl, rat growth hormone cDNA, human LeIF_(D),hepatitis B surface antigen) ADHl transcription starting specificity istransplanted and expression is promoted. While the requirements fortranscription initiation are easily met, expression at the protein levelis not observed in all cases". Differences in results were generallyattributed to variations in "posttranscriptional events" of variouspossible types.

There thus continues to be a need in the art for yeast celltransformation vectors useful in securing the high level expression inyeast host cells of exogenous genes coding for useful proteins.

C. Hepatitis B Surface Antigen

Of particular interest to the background of the novel preparations ofthe present inventions which display the immunological activity ofhepatitis B surface antigen are the numerous publications and patentstreating the nature of the hepatitis B viral disease state as well asmethods and materials developed for preparing vaccines protective ofsusceptible mammals against viral infection.

Hepatitis B virus constitutes a public health problem of enormousproportions affecting some 2 million persons worldwide. In the U.S.alone, some 100,000 to 200,000 cases of hepatitis are annuallyattributed to infection by this virus and it is reliably estimated thatsuch infection results in nearly 2,000 fatalities each year. A majorstep in the prophylaxis against hepatitis B infection was the 1965discovery of various circulating antigen particles in the serum ofinfected mammals, including humans. In the early 1970's, it wasestablished that subunit vaccines comprising hepatitis B surface antigenpreparations isolated from the plasma of chronic carriers could providea protective immune response against infection by the virus. Since thattime, enormous research and developmental effort has been expended withthe goal of optimizing the procedures by which the surface antigen(HBsAg) could be isolated in quantity and incorporated into effectivevaccine compositions. Reflecting this effort are the numerous U.S.Letters Patent in this subject area which have issued in the last fiveyears alone, including U.S. Pat. Nos. 4,024,243; 4,057,628; 4,113,712;4,017,360; 4,118,477; 4,118,478; 4,129,646; 4,138,287; 4,186,193;4,164,565; and U.S. Pat. No. 4,242,324.

A hepatitis B vaccine composition very recently made available is theproduct marketed by Merck, Sharp and Dohme, Inc. as "Heptavax-B". Thisproduct is recommended for immunization against infection caused by allknown subtypes of hepatitis B virus in persons 3 months of age or older.Each 1.0 ml dose of the vaccine contains 20 μg of hepatitis B surfaceantigen formulated in an alum adjuvant. The immunization regimenconsists of 3 doses of vaccine given intramuscularly, with dose volumesranging from 0.5 to 2.0 ml according to the age, weight or othercharacteristics of the vaccinate.

The specific antigen preparation employed in the above-noted vaccineconsists of purified, antigenic spherical particles of an averagedimension of about 22 nanometers (nm) which are isolated from the plasmaof chronic carriers by such exhaustive purification methods asfractional precipitation, chromatographic separation, sequentialisopycnic and rate zonal centrifugation, peptic digestion and variouschemical treatments.

Upon isolation from plasma, the active, HBsAg subunit vaccine particlepreparations are characterized by a lipoproteinaceous constitutionwherein the major phospholipids are identified as phosphotidylcholineand sphingomyelin. There has also been found evidence of carbohydrate inHBsAg preparations in the form of glycoproteins and/or glycolipids.Degradative analysis of the protein moiety of the HBsAg preparationspurified from plasma has revealed mixtures of polypeptides ranging inmolecular weight from 19,000 to 120,000 daltons.

As a result of the above-noted physical characteristics, the 22 nm HBsAgparticle is frequently visualized as comprising disulfide-bonded pairsof identical polypeptide strands (each of about 25,000 daltons) havinglipids associated with and shrouding hydrophobic amino acid rich regionsand also possibly having carbohydrates linked to exposed hydrophilicamino acid-rich regions which generally "present" short, antigenicpolypeptide sequences to the host.

The HBsAg in the lipid envelope has one well-characterized groupspecific determinant, a, and two sets of mutually exclusive subtypedeterminants, d/y and w/r. Four major subtypes of HBsAg exist: adw, ayr,ayw, and adr. Other intermediate specificities have led to theclassification of HBsAg into ten serological categories.

Numerous attempts have been made to dispositively ascertain the aminoacid sequence of the major protein(s) of HBsAg and the nucleotidesequence of the viral gene coding for the protein. For example,Valenzuela, et al. Nature, 280, pp. 815-819 (1979) treats the extractionof DNA from hepatitis B viral particles and the cloning and isolation ofan 892 base pair fragment believed to encode for the proposed majorprotein component of HBsAg. Based on nucleotide base sequence analysis,a 226 amino acid polypeptide sequence was deduced which preciselycorresponds in its initial N-terminal region to published amino acidsequences for that region ascertained by peptide degradative analysis.The published amino acid sequence is as follows: ##STR1##

The above-noted work directed toward amino acid analysis of polypeptideconstituents of HBsAg preparations has been of significant value tothose seeking to develop synthetic vaccines, i.e., "second generation"hepatitis B polypeptide vaccines containing hepatitis B-specificantigenic components. See, e.g., Dreesman, et al., Nature, 295, pp.158-160 (1982), Edman, et al., Nature, 291, pp. 503-506 (1981) andZuckerman, Nature, 295, pp. 98-99 (1982). In the Dreesman, et al.publication, it was reported that synthetic peptide sequences of 16 to21 amino acids duplicative of putative HBsAg sequences appeared to becapable of stimulating antibody production responses in host animalseven without "mounting" of the sequences on carrier particles. Noimmunoprotective properties were attributed to the sequences, however.In the Edman, et al. publication, work on bacterial synthesis of HBsAgsequences as fusion polypeptides is reported. These polypeptidesappeared to include one or more antigenic determinations found in nativeHBsAg and, while investigation into immunogenic characteristics wasprojected, immunoprotective properties on par with carrier-derived 22 nmparticles was not expected to be obtained. In the Zuckerman publication,it was reported that, "Expression of glycosylated hepatitis B surfaceantigen in yeast cells has recently been reported by W. J. Rutter of theUniversity of California, San Francisco, and this is potentially a mostimportant development for large-scale in vitro production of animmunogen". The text, however, provides no particulars as to thephysical nature of the glycoprotein antigen, its immunologicalcharacteristics, or the manner and quantities in which it may have beenobtained.

Only very recently has any detailed report been published concerning thework on HBsAg announced by Zuckerman, supra. Specifically, an article byValenzuela, et al. appearing in Nature, 298, pp. 347-350 (July 22, 1982)reports on the synthesis and assembly of Hepatitis B virus surfaceantigen particles in yeast. Briefly summarized, the HBsAg antigen wasnoted to have been synthesized in yeast using an expression vector thatemployed the 5' flanking region of yeast alcohol dehydrogenase I as apromoter to transcribe surface antigen coding sequences. The proteinsynthesized in yeast was said to have been assembled into particleshaving properties similar to the 22 nm particles secreted by humancells. The publication of Valenzuela, et al., Nature, 298, pp. 347-350(1982) is specifically incorporated herein for purposes of providinginformation relevant to the background of the present invention.

While significant advances appear to have been made toward theprocurement of immunologically active HBsAg preparations from sourcesother than human carrier plasma, prior to the present invention the arthad not been provided with certain means for obtaining such preparationsin significant quantity, nor had the fragmentary reports of successes inthis area been accompanied by any showing of useful immunoprotectiveproperties.

BRIEF SUMMARY

In one of its aspects, the present invention provides a new class oftransformation vectors which facilitate the high level expression inyeast cells of genes which are exogenous to yeast and which code forcommercially useful polypeptides. More specifically provided are yeastcell transformation vectors comprising: (1) a DNA transcriptionpromoter/regulator DNA sequence endogenous to yeast cell synthesis ofmRNA coding for glyceraldehyde-3-phosphate dehydrogenase; (2) a DNAsequence transcribable into mRNA coding for synthesis of a polypeptideexogenous to yeast cells; (3) a DNA sequence coding for termination oftranscription of and/or poly-A addition to mRNA in yeast cells; and (4)a DNA sequence operative in yeast cells to confer to said vector thecapacity for autonomous DNA replication. The vectors may also desirablycontain DNA sequences which allow selection of yeast cells containingthe vectors.

In preferred embodiments of vectors constructed according to theinvention, the promoter/regulator region substantially duplicatesportions of the putative promoter/regulator region of aglyceraldehydge-3-phosphate dehydrogenase (G-3-PDH) gene of yeast cellline Saccharomyces cerevisiae S288C. This DNA fragment can be isolatedas an approximately 680 base pair fragment by TaqI restrictionendonuclease enzyme digestion of a cloned 2.1 kilobase Hind III fragmentcontaining the G-3-PDH gene. Preferred embodiments of vectors alsoinclude, e.g., a 2μ replicon or autonomous replicating sequences such asARS-1 and may also have mRNA transcription termination and/or poly-Aaddition sequences provided as a portion of a TRPl gene located 3' andin the same polarity to the exogenous gene whose expression is sought.When an entire TRPl gene is employed to supply transcription terminationand/or poly-A addition sequences, it may serve as a selectable markerfor yeast transformation and vector maintenance. The 3' ends of otheryeast genes which include sequences responsible for transcriptiontermination and/or poly-A addition may also be utilized.

Because the manufacture of yeast transformation vector of the inventionordinarily involves carrying out manipulations of DNA sequences inbacterial cells, the vectors may retain some or all of the requisiteproperties of "shuttle" vectors, i.e., DNA sequences providing forautonomous replication and selectable transformation in bacteria.

Also comprehended by the invention are transformation procedures forsecuring the expression, in yeast cells, of exogenous polypeptidesequences and for forming novel isolatable proteinaceous productsincluding not only polypeptides (proteins) but also lipoproteins,glycoproteins and glycolipoproteins. Such products are unique in termsof their in vitro origins in yeast cells (rather than the life forms towhich they are endogenous) and may also be uniquely characterized by thepresence of carbohydrate and/or lipid constituents peculiar to theiryeast cell origins.

DNA sequences transcribable into mRNA for exogenous polypeptides mayderive from a wide variety of life forms including procaryotic andeucaryotic cells and viruses. The most commercially significant DNAsequences for incorporation into vectors of the invention are ofeucaryotic origin and, consistent with prior studies of yeastexpression, preferably are free of introns. DNA sequences may be clonedgenomic isolates, cDNA's or totally or partially manufactured DNAsequences.

Illustrative of an exogenous DNA sequence constituent of vectors of theinvention is a DNA sequence transcribable into mRNA coding forsynthesis, in yeast, of a polypeptide having one or more of theimmunological activities of HBsAg. Also illustrated are DNA sequenceswhich comprise a series of base codons duplicative of codons endogenousto the HBsAg gene in hepatitis B viral genome and a manufacturedsequence of base codons selected on the basis of optimal yeast codonpreferences.

In another of its aspects, the present invention provides novelpreparations possessing one or more of the immunological activities ofHBsAg, which preparations may suitably be employed in the manufacture ofvaccine compositions. The vaccine compositions so manufactured may beemployed in the manner of commercial HBsAg vaccines to provoke theformation of antibodies protective against hepatitis B virus infectionof a susceptible mammalian species vaccinate. Additionally, HBsAgpreparations of the invention are expected to be quite usefully employedas reagents in procedures for quantitative detection of hepatitis Bvirus associated markers in fluid samples.

The novel HBsAg preparations of the present invention includeproteinaceous materials isolated from genetically transformed yeastcells, which materials (a) respond positively in standardizedradioimmunoassays or enzyme linked immunoassays for HBsAg of serumorigin; (b) are immunologically neutralized by human antibodies toHBsAg; (c) sediment at greater than 28 S in sucrose gradients; and (d)have a buoyant density in cesium chloride which is somewhat less thanthat of HBsAg particles of virally-infected human serum origin (i.e.,less than 1.210). These proteinaceous yeast cell products are thusbelieved to be lipoproteins which may also be glycosylated and appear tohave a spherical particulate configuration.

A DNA vector, pHBs-1[GPD] suitable for use in practice of the inventionto stably transform yeast cells and to thereby secure the expression ofreadily isolatable quantities of a lipoprotein preparation having one ormore of the immunological activities of HBsAg of viral origin wasdeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Maryland, 20852 on Aug. 20, 1982, in accordance withthe Patent and Trademark Office's requirements for microorganismdeposits, and was designated as A.T.C.C. No. 40053.

Further aspects and advantages of the present invention will be apparentupon considerations of the following detailed description of thepractice of preferred embodiments thereof, reference being made to FIGS.1 through 4 which provide schematic illustrations of certain geneticmanipulations performed according to the invention and certain DNAsequences involved in these manipulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of pHBs-1[GPD[.

FIG. 2 shows a restriction map of part of pHBs-1[GPD].

FIG. 3 shows the construction of p(GPD-BGs)-2.

FIG. 4 shows the construction of p(GPD-HBs)-3.

DETAILED DESCRIPTION

The following examples illustrate practice of the invention in theconstruction of illustrative yeast cell transformation vectors,including pHBs-1[GPD] [A.T.C.C. No. 40053] and in the preparation andcharacterization of novel lipoprotein products of yeast origin whichpossess immunological activities ordinarily displayed by HBsAg of viralorigin. More particularly, Examples 1 through 4 are directed to thefollowing manipulations performed with respect to plasmid pHBsAgcomprising the shuttle vector, YRp7 (pRB16, A.T.C.C. 37052), into whichhad been cloned a 1350 base pair genomic DNA fragment of hepatitis Borigin including a HBsAg gene; deletion from pHBsAg of HBV DNA sequences3' to the HBsAg coding region; isolation of a putativepromoter/regulator DNA sequence fragment from a G-3-PDH gene of yeastcell line Saccharomyces cerevisiae S288C; insertion of thepromoter/regulator DNA sequence 5' to the HBsAg gene in pHBsAg to formplasmid pHBs-1[GPD]; and transformation of yeast cells with thepHBs-1[GPD]. Examples 5 through 9 relate to characterization of novellipoprotein HBsAg products of yeast origin in term of their physicalproperties and immunological activities. Also provided are constructionsof additional transformation vectors which include DNA sequences codingfor HBsAg synthesis in yeast.

DNA sequences according to the present invention may also bemanufactured using one or more of the "optimal" yeast preference codonsset forth in Table I. [See Bennetzen, et. al., J. Biol. Chem., 257,3026-3031, (1982)]

                  TABLE I                                                         ______________________________________                                        Amino Acid        "Optimal" Codon                                             ______________________________________                                        Ala               5'-GCT or 5'-GCC                                                              CGA CGG                                                     Arg               5'-AGA                                                                        TCT                                                         Asp               5'-GAC                                                                        CTG                                                         Asn               5'-AAC                                                                        TTG                                                         Cys               5'-TGT                                                                        ACA                                                         Gln               5'-CAA                                                                        GTT                                                         Glu               5'-GAA                                                                        CTT                                                         Gly               5'-GGT                                                                        CCA                                                         His               5'-CAC                                                                        GTG                                                         Ile               5'-ATT or 5'-ATC                                                              TAA TAG                                                     Leu               5'-TTG                                                                        AAC                                                         Lys               5'-AAG                                                                        TTC                                                         Phe               5'-TTC                                                                        AAG                                                         Pro               5'-CCA                                                                        GGT                                                         Thr               5'-ACT or 5'-ACC                                                              TGA TGG                                                     Tyr               5'-TAC                                                                        ATG                                                         Ser               5'-TCT or 5'-TCC                                                              AGA AGG                                                     Val               5'-GTT or 5'-GTC                                                              CAA CAG                                                     ______________________________________                                    

EXAMPLE 1

A DNA plasmid designated pHBsAg was obtained from collaborators atAbbott Laboratories, North Chicago, Illinois. The plasmid was reportedto have been constructed by manipulations performed on a yeast-bacteriashuttle vector, YRp7 (A.T.C.C. 37052). More specifically, in a manneranalogous to that illustrated for preparation of the bacterial plasmidpHBV-3200 from pBR322 by Valenzuela, et al., supra, a 1350 base pair DNAfragment obtained by BamHI treatment of a cloned hepatitis B genome wasinserted at the BamHI site of YRp7 using T4 DNA ligase. The fragment wasinserted in the same polarity as the TRP1 gene of YRp7 at the BamHI siteabout 400 base pairs 5' to the TRPl gene. The resultant vector wasamplified by transformation of E. coli accompanied by selection based onthe presence of the AMP gene marker native to YRp7.

Upon receipt of the plasmid, partial DNA sequence analysis andrestriction enzyme mapping of the inserted fragment was conducted andrevealed (reading 5' to 3'): (1) the 5' end BamHI cleavage site occurs126 base pairs 5' to the first ATG codon (transcribable to an AUG mRNAtranslation initiation codon) in the sequence; (2) an XbaI recognitionsite mapped by restriction enzyme digestion exists in the polypeptidecoding region commencing about 94 base pairs 3' to the above-noted ATGcodon and about 218 base pairs 3' to the BamHI cleavage site; (3) aunique HpaI recognition site mapped by restriction enzyme digestionexists approximately 715 base pairs 3' to the XbaI recognition site and933 base pairs 3' to the BamHI cleavage site; and (4) the 3' BamHIcleavage site is approximately 440 base pairs 3' to the HpaI cleavagesite. Observed characteristics (1) and (2), above, are in accord withthe DNA sequence published by Valenzuela, et al., supra. Specificsequencing of the top strand (5' to 3') revealed the following as theidentities of the first 143 bases of the HBV fragment: ##STR2## Exceptthat a thymine rather than cytosine was identified at the sitedesignated by an asterisk, the sequence is in complete accord with thatof Valenzuela, et al., supra. The HBsAg gene insert, which was clonedfrom pooled sera, is presumably of serotype adw 2 reported byValenzuela, et al.. It is noteworthy that this fragment includes anadenine base at a position "-3" to the ATG. This is a feature believedto be present in most endogenous yeast structural genes and which hasbeen implicated as significant to gene expression.

EXAMPLE 2

Plasmid pHBsAg was reconstructed by deletion of certain DNA sequencesintermediate the HBsAg gene and the TRPl gene as follows. The plasmidwas completely digested with HpaI restriction endonuclease. Thelinearized product was then partially digested using Hind III resultingin formation of a mixture of linear fragments which differed in lengthdepending on which of the two Hind III recognition sites provided thecleavage site. The approximately 6370 base pair fragment (which retainedthe Hind III recognition site within the yeast TRPl gene but was cleavedat the Hind III site of pBR322) was purified. The cohesive ends of thefragment were then filled in (i.e., rendered "blunt") with DNAPolymerase I large fragment (Klenow fragment) (Bethesda Research Labs.)and the plasmid religated using T4 ligase. The resulting circularizedDNA plasmid, pHBs-1, was amplified and purified through transformationof E. coli accompanied by selection based on the presence of the AMPgene marker. The net result of the procedure was the elimination of anapproximately 780 base pair fragment of both HBV and pBR322 origin,bringing the TRPl gene into close proximity to, and in the same polaritywith, the HBsAg gene. This projectedly facilitates yeast RNA polymeraseII transcription of the TRPl gene by "read-through" transcription fromthe surface antigen gene. This, in turn, functionally associates suchtranscription termination and/or poly-A addition sequences as are extantin the yeast TRPl gene with the HBsAg gene so that transcriptionalevents attending HBsAg mRNA formation based on vector DNA closelysimulate those of endogenous genes. Additionally, plasmid pHBs-1contains a unique BamHl site 126 bp upstream from the ATG initiator ofthe surface antigen gene. There are no ATG sequences in the codingstrand between the BamHI site and the ATG initiator of the surface gene.Thus, this unique BamHI site may be used for cloning and functionaltesting of putative yeast promoter fragments.

EXAMPLE 3

A yeast glyceraldehyde-3-phosphate dehydrogenase gene DNA fragment neverbefore shown to be a promoter was isolated for use as apromoter/regulator sequence in vectors of the invention according to thefollowing procedure. A 2.1 kilobase yeast Hind III fragment containingthe G-3-PDH gene isolated from a phage lambda yeast DNA library of yeastcell line S288C, was subcloned in pBR322. (Dr. R. A. Kramer, Nat'l.Cancer Inst., Nat'l. Institute of Health, Bethesda, Md., Musti, et al.,1981 Cold Spring Harbor Meeting, Abstract No. 215, Cold Spring HarborLaboratory; see also, Holland, et al., J. Biol. Chem., 254, pp.9839-9845 (1979)). This plasmid was digested to completion with Hind IIIand an approximately 2100 base pair DNA fragment was isolated. Thisfragment was then digested to completion using TaqI and the largestfragment (approximately 680 base pairs) was purified. Based on priorpublications of results of the sequencing of the 5' untranslated regionof the yeast G-3-PDH gene, the isolated fragment did not include the 26base pair sequence ##STR3## present in the endogenous gene prior to theinitial ATG of the structural gene. The DNA fragment used herein as apromoter is the 680 base pair fragment upstream (5' to the codingregion) of the 26 base pair sequence noted.

EXAMPLE 4

Plasmid pHBs-1[GPD], A.T.C.C. 40053 was constructed as follows. Copiesof plasmid pHBs-1 according to Example 2 were digested to completionwith BamHl and mixed with the 680 base pair fragment of Example 3.Cohesive ends were made blunt using DNA Polymerase I large fragment(Bethesda Research Labs), the fragments were ligated with T4 DNA ligase,and E. coli clones which contained a single 680 base pair fragmentinserted in the correct orientation were isolated. The construction ofplasmid pHBs-1[GPD] and its structure are illustrated in pertinent partin FIGS. 1 and 2. The vector is seen to include a putative DNAtranscription promoter/regulator DNA sequence endogenous to yeast cellsynthesis of mRNA coding for glyceraldehyde-3-phosphate dehydrogenase.This approximately 680 base pair sequence is derived from andsubstantially duplicates a significant portion of the promoter/regulatorregion of the G-3-PDH gene of yeast cell line Saccharomyces cerevisiaeS288C. Following this endogenous yeast sequence is an approximately 126base pair sequence of viral origin which substantially duplicates theuntranslated leader region preceding the initial ATG codon of a HBsAggene in the hepatitis B viral genome. The vector then includes a DNAsequence transcribable into mRNA coding for the synthesis of apolypeptide exogenous to yeast (i.e., a polypeptide-specifying HBsAggene) in the same polarity a the promoter/regulator. Approximately 29base pairs of pBR322 DNA separates the antigen gene from a completeyeast TRP1 gene which includes a base sequence coding for transcriptiontermination of and/or poly-A addition to mRNA in yeast. Finally, thevector includes a DNA sequence (i.e., the ARS-1 sequence) operative inyeast cells to confer upon the vector the capacity for autonomousreplication.

EXAMPLE 5

Yeast Saccharomyces cervisiae RH218 cells were transformed with thepHBs-1[GPD] vectors of Example 4 according to the procedure of Hinnen,et al., Proc. Nat'l. Acad. Sci. U.S.A., 75, 1929-1933 (1978) withtransformants selected on the basis of expression of the TRPl gene bygrowth of transformed host cells in minimal media lacking tryptophan.Whole cell protein extracts of transformed cells were prepared byconverting transformed yeast cells to spheroplasts with Zymolase 60,000(Kirin Breweries, Japan) in the presence of the osmotic stabilizer, 0.9M sorbitol. The spheroplasts were collected by centrifugation and lysedby resuspending in 3 volumes of 25 mM Tris-HCl, pH 7.5. (In someinstances, the whole cell lysate was sonicated.) The whole cell lysatewas subjected to low-speed centrifugation and the supernatant utilizedas the source of yeast-produced HBsAg.

EXAMPLE 6

Whole cell protein extracts of yeast cells transformed according toExample 5 and cultured in the absence of tryptophane were assayed forhepatitis B surface antigen using the Auszyme II Immunoassay kit (AbbottLaboratories, North Chicago, Illinois). Briefly summarized, theprocedure provides for quantitative detection of HBsAg in a fluid sampleby a "sandwich assay". Beads which are coated with antibody to HBsAg areincubated with a solution which may contain HBsAg. Any HBsAg in thesolution will bind to the antibody on the bead. After washing, the beadsare incubated with a second solution of antibody to HBsAg. This secondantibody is linked to horseradish peroxidase, and will bind to antigenwhich may have bound to the first antibody on the bead. After washing,the amount of HBsAg bound to the bead is quantitated by assaying theamount of horseradish peroxidase (and hence, second antibody) bound tothe bead.

When extracts were prepared from untransformed Saccharomyces cerevisiaeRH218, no HBsAg reactive material was detected by the above assay.However, when extracts were prepared from the same cells transformedwith recombinant plasmid pHBs-1[GPD], HBsAg reactive material wasdetected. Furthermore, this reactive material exhibits a clear doseresponse with increasing yeast protein concentration in the Auszyme IIreactions.

EXAMPLE 7

To provide confirmation that the immunological activity displayed bywhole yeast cell extracts in Example 6 was indeed duplicative of HBsAgof viral origins, tests were conducted to determine whether suchactivity would be "blocked" or neutralized by pre-incubation with humananti-HBsAg antibody as is characteristic of HBsAg positive human serumsamples.

This procedure (Auszyme II Confirmatory Neutralization Test, AbbottLabs.) adds an additional step to the procedure of the Auszyme II assaydescribed in Example 6. Before the addition of the second antibody, thebead is incubated with either negative human serum or human antibody toHBsAg. If a material which gives positive results in the Auszyme IIassay is HBsAg, it should react with human antibody to HBsAg andtherefore be unable to react with the horseradish peroxidase-linkedsecond antibody. The immunoreaction will have been "neutralized". As acontrol, HBsAg immunoreactivity should not be effected by anintermediate incubation with negative human serum.

The results of the test procedure are summarized in Table I below andreveal that the Auszyme II reactive material in the preparations ofyeast origin is substantially duplicative of the HBsAg antigen in thepositive human serum which is commonly employed as a source ofingredients for commercial subunit vaccines.

                  TABLE II                                                        ______________________________________                                                      INTERMEDIATE     A492                                           SAMPLE        INCUBATION       VALUES                                         ______________________________________                                        Negative Human Serum                                                                        Negative Human Serum                                                                           0.027                                          Negative Human Serum                                                                        Negative Human Serum                                                                           0.014                                          Negative Human Serum                                                                        Negative Human Serum                                                                           0.012                                          Positive Human Serum                                                                        Negative Human Serum                                                                           2.560                                          Positive Human Serum                                                                        Negative Human Serum                                                                           2.770                                          Positive Human Serum                                                                        Human Anti-HBsAg 0.053                                          Positive Human Serum                                                                        Human Anti-HBsAg 0.030                                          Yeast Extract Negative Human Serum                                                                           0.808                                          Yeast Extract Negative Human Serum                                                                           0.771                                          Yeast Extract Human Anti-HBsAg 0.025                                          Yeast Extract Human Anti-HBsAg 0.028                                          ______________________________________                                    

EXAMPLE 8

A gross estimate of the structural size of the yeast-produced HBsAg canbe determined by its rate of sedimentation in sucrose gradients.

Yeast whole cell extracts are mixed with Tritium-labelled ribosomal RNAand centrifuged in 5-40% sucrose gradients. Fractions are collected, andsedimentation of the ribosomal RNA is measured by determiningradioactivity in individual fractions. Sedimentation of the yeast HBsAgwas monitored by assaying fractions with the Auszyme II test (Example6).

Soluble monomeric protein of the size of HBsAg is expected to sedimentat about 2S-3S. The yeast HBsAg sedimented at greater than 28S,indicating aggregation into a larger particle structure.

EXAMPLE 9

The similarities and differences between yeast-produced HBsAgpreparations of the invention and HBsAg preparations of human origin aspresently employed in commercial vaccines are exemplified by buoyantdensity characteristics of the two substances in cesium chloride.

Solid cesium chloride is added to the yeast whole cell extract and topositive human serum to give a final density of 1.200 gm/cc to eachsolution. The solutions are centrifuged to equilibrium and fractions arecollected. For each fraction, the density is determined and the amountof HBsAg measured using the Auszyme II test (Example 6).

The buoyant density of the HBsAg in human serum was determined to beapproximately 1.210 gm/cc while the yeast origin HBsAg preparations hada buoyant density of 1.196 gm/cc. These results, when considered inlight of the previously-noted immunological activity determinations,direct the following conclusions. The yeast-produced HBsAg preparationsof the invention possess significant immunological properties which arecharacteristic of antigen preparations obtained from human carrierserum. The preparations are nonetheless physically distinct substancespresumably owing to the presence of yeast origin lipids rather thanhuman origin lipids or some difference in the spectrum of proteinspresent in the particle.

Purification procedures to be employed to isolate lipid particlescontaining HBsAg from transformed yeast cells include rate zonalcentrifugation, repeated isopycnic centrifugation, fractionalprecipitation and chromatographic techniques.

Vaccine compositions according to the present invention may beformulated in the same manner as noted above with regard to commercialvaccines incorporating viral origin HBsAg isolates from human serum.Unit doses of 1.0 ml volume may be formulated containing approximately20μg of antigen isolated from yeast and may include standardimmunologically acceptable diluents, adjuvants and carriers. In a likemanner, vaccination procedures of the present invention for the purposeof provoking a protective immune response in a susceptible mammalcorrespond to those practiced using HBsAg vaccine preparations. Anexemplary immunization regimen may thus consist of 3 IM doses of from0.5 to 2.0 ml of vaccine.

The foregoing illustrative examples are particularly directed todescription of the manufacture of the specific yeast transformationvector, pHBs-1[GPD], A.T.C.C. No. 40053, and to the use of the vector inyeast transformation resulting in expression of a specific proteinaceousmaterial, HBsAg. The scope of the present invention is clearly notlimited, however, to the particular vector manufactured or theparticular proteinaceous products produced.

With respect to the specific G-3-PDH promoter/regulator DNA sequenceincorporated in pHBs-1[GPD], it is expected that equivalent results inthe promotion and regulation of transcription of exogenous DNA willattend the use of DNA fragments of at least somewhat smaller size thanthe 680 base pair fragment of the examples without significant decreasesin levels of expression. In a like manner, it is not unlikely thatfurther reconstruction of pHBs-1[GPD] to restore the approximately 25base pairs present in the endogenous yeast sequence and/or to delete theapproximately 126 base pairs intermediate the exogenous polypeptidecoding region and the promoter/regulator sequence will result inenhancement of expression. Such a procedure would involve synthesizingan oligonucleotide as a BamHI to XbaI fragment. The sequences adjacentto the BamHI site would include the untranslated G-3-PDH leader sequencenot present in the 680 bp TaqI fragment (i.e., the 26 bp sequenceillustrated in Example 3). After the ATG, the first 32 codons of thesurface antigen gene would be resynthesized with codons known to bepreferentially, optimally or highly frequently utilized in yeast(Grantham, et al., Nucleic Acids Research, 8, pp. r49-r62 (1980);Grantham, et al., Nucleic Acids Research, 8, pp. 1893-1912 (1980); and,Grantham, et al., Nucleic Acids Research, 9, pp. r43-r74 (1981).

Illustrative of such a sequence is the following: ##STR4## Alternativesequences which could suitably be employed would provide: theisoleucine-specifying codons as ##STR5## the threonine-specifying codonsas the serine-specifying codons as the valine-specifying codons as andthe alanine-specifying codons as Each of these potential replacementswould be consistent with predominant patterns of yeast codon usage forhighly expressed endogenous yeast genes. In general, construction of awholly manufactured sequence comprising all or part of an exogenous geneto be expressed in yeast according to the invention would avoid, to theextent consistent with manufactured sequence manipulations, usage of##STR6##

Such fragments would then be cloned into pHBs-1 and the G-3-PDHpromoter/regulator fragment would substantially be cloned into the BamHIsite. In a like manner, the remainder of the HBsAg gene could beresynthesized using optimal yeast codons. This would be expected toresult in expression levels of HBsAg in yeast comparable to those ofG-3-PDH (1-5% of cellular protein). Such a synthetic approach wouldallow for synthesis in yeast of HBsAg of all serotypes, providing forvaccines protective against all known serotypes of Hepatitis B.

It is clearly the case that numerous DNA sequences coding for exogenouspolypeptide may be substituted for the HBsAg polypeptide coding sequencepresent in pHBsl[GPD] to provide for high level expression ofcorresponding proteinaceous materials by transformed yeast cells.[Indeed, it is contemplated that active HBsAg preparations may beobtained through use of vectors including DNA sequences coding forrelatively short polypeptides possessing the immunological activity of22 nm particles.] In keeping with the results and proposals of priorinvestigations, it is expected that highest levels of expression willattend the use of intronfree DNA sequences which incorporate, to thegreatest degree possible, alternative codons of the type preferentiallyexpressed in yeast cells.

In the foregoing illustrative examples, a transcription terminationand/or poly-A addition sequence was supplied in close proximity to theexogenous gene to be expressed in the form of an entire yeast TRPl geneand this gene also served as a selectable marker for yeasttransformation. Clearly transcription termination and/or poly-A additionsequences may be derived from 3' untranslated regions of yeast genes andsupplied to vectors of the invention independently of incorporation ofan entire structural gene. In a like manner, marker genes other thanTRPl can be employed if desired.

While the ARSl origin of replication was successfully employed to donateautonomous replicative capacity to pHBs-l[GPD] in the foregoingillustrative examples, functionally equivalent origin of replicationsequences such as a part or all of the 2μ replicon may be employed. Forexample, two vectors were recently prepared using an approximately 2800base pair SalI to BglII fragment of pHBs-l[GPD] which included the yeastG-3-PDH promoter/regulator region, the HBsAg gene and the entire yeastTRP-1 gene (along with a small DNA sequence of pBR322 origin) but notthe ARS 1 segment. Construction of such vectors is illustrated in FIGS.3 and 4. In a first construction, this 2800 base pair fragment wasinserted into the large fragment obtained by SalI and BamHI digestion ofplasmid pGT40 to provide plasmid p(GPD-HBs)-2 including the Ori and Rep2 sequences (between adjacent EcoRI sites) but not the Rep 1 sequence.In a second construction, the 2800 base pair fragment was inserted intothe large fragment obtained by SalI and BamIH digestion of plasmid pGT41to provide plasmid p(GPD-HBs)-3 including the Ori and both Rep 1 and Rep2 sequences. Plasmid p(GPD-HBs)-2 is expected to be suitable for use intransformation of Cir⁺ yeast strains while p(GPD-HBs)-3 can be employedto transform Cir^(O) strains (having no endogenous 2μ plasmid).Preliminary studies have shown an approximately 20 to 50 foldimprovement in levels of expression of HBsAg by yeast cells transformedwith p(GPD-HBs)-3 as opposed to pHBs-l[GPD].

Numerous modifications and variations in the invention are expected tooccur to those skilled in the art upon consideration of the foregoingdescription. Consequently, only such limitations as appear in theappended claims should be placed on the invention.

What is claimed is:
 1. A yeast cell transformation vector comprising:(1) a DNA transcription promoter/regulator DNA sequence duplicative ofthat endogenous to yeast cell synthesis of mRNA coding forglyceraldehyde-3-phosphate dehydrogenase wherein said DNA does notinclude the 26 base pairs having the sequence ##STR7## (2) a DNAsequence transcribable into mRNA coding for synthesis of a polypeptideexogenous to yeast cells; (3) a DNA sequence duplicative of that codingfor termination of transcription or poly-A addition of mRNA in yeastcells; and (4) a DNA sequence operative n yeast cells to confer to saidvector the capacity for autonomous DNA replication wherein said yeast isSaccharomyces cerevisiae.
 2. A yeast cell transformation vectoraccording to claim 1 wherein said DNA sequence transcribable into mRNAcoding for synthesis of a polypeptide exogenous to yeast istranscribable into mRNA coding for synthesis of a polypeptide having heimmunological activity of HBsAg.
 3. A yeast cell transformation vectoraccording to claim 2 wherein the polypeptide coded for has theimmunological activity of ADW2 serotype HBsAg.
 4. A yeast celltransformation vector according to claim 3 wherein the polypeptide codedfor has an amino acid sequence substantially comprising: ##STR8##
 5. Ayeast cell transformation vector according to claim 1 wherein said DNAsequence coding for termination of transcription and/or poly-A additionof mRNA is a sequence substantially duplicative of a sequencefunctionally associated with a TRP1 gene of yeast origin.
 6. A yeastcell transformation vector according to claim 1 wherein said DNAsequence operative to confer autonomous DNA replication is substantiallyduplicative of a yeast DNA replication origin sequence selected from thegroup consisting of ARS and 2μ replicon sequences.
 7. A yeast celltransformation vector according to claim 1 comprising (1) a DNAtranscription promoter/regulator DNA sequence duplicative of thatendogenous to yeast cell synthesis of mRNA coding forglyceraldehyde-3-phosphate dehydrogenase wherein said DNA does notinclude the 26 base pairs having the sequence ##STR9## (2) a DNAsequence transcribable into mRNA coding for synthesis of a polypeptideexogenous to yeast cells; (3) a DNA sequence duplicative of that codingfor termination of transcription and poly-A addition of mRNA in yeastcells; and (4) a DNA sequence operative in yeast cells to confer to saidvector the capacity for autonomous DNA replication.
 8. A yeast celltransformation vector according to claim 1 wherein said transcriptionpromoter/regulator DNA sequence is substantially duplicative of anapproximately 680 base pair DNA sequence derived by TaqI restrictionendonuclease enzyme digestion of a 2.1 kilobase Hind III DNA fragment ofSaccharomyces cerevisiae S288C origin containing a yeastglyceraldehyde-3-phosphate dehydrogenase gene wherein said DNa does notinclude the 26 base pairs having the sequence ##STR10##
 9. PlasmidHBs-1[GPO], A.T.C.C. No.
 40053. 10. A yeast cell transformation vectorcapable of autonomous replication in yeast and useful in securingexpression by transformed yeast cells of a polypeptide having theimmunological activity of HBsAg, said vector comprising (1) a DNAtranscription promoter/regulator DNA sequence duplicative of thatendogenous to yeast cell synthesis of mRNA coding forglyceraldehyde-3-phosphate dehydrogenase wherein said DNA does notinclude the 26 base pairs having the sequence ##STR11## and (2) anexogenous DNa sequence transcribable into mRNA coding for synthesis of apolypeptide having the immunological activity of HBsAg wherein saidyeast is Saccharomyces cerevisiae.